Corneal epithelial and retinal pigment epithelial cell culture assays as potential alternatives to animal experimentation for the evaluation of ocular toxicity

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dc.contributor Aalto-yliopisto fi
dc.contributor Aalto University en
dc.contributor.advisor Uusitalo, Hannu; Prof.
dc.contributor.advisor Tähti, Hanna; Prof.
dc.contributor.author Huhtala, Anne
dc.date.accessioned 2011-12-20T10:44:52Z
dc.date.available 2011-12-20T10:44:52Z
dc.date.issued 2003-10-10
dc.identifier.isbn 951-22-6734-9
dc.identifier.issn 0359-6621
dc.identifier.uri https://aaltodoc.aalto.fi/handle/123456789/1210
dc.description Väitöstutkimuksessa kartoitettiin eläinkokeille vaihtoehtoisia menetelmiä, joilla voitaisiin korvata kanin silmä-ärsytystesti, ns. Draizen testi. Draizen testi on ainoa maailmanlaajuisesti viranomaiskäyttöön hyväksytty toksisuustesti, jota käytetään arvioitaessa kemikaalien aiheuttamaa silmä-ärsytystä. OECD:n ohjeista puuttuu myös testi, jolla voitaisiin arvioida verkkokalvoon kohdistuvia haittoja. Kokeellisessa tutkimuksessa kehitettiin silmän uloimpaan osaan, sarveiskalvon soluihin ja silmän näkevään osaan, verkkokalvon soluihin perustuvia testejä käytettäviksi toksisuuden arvioinnissa osana laajempaa eläinkokeille vaihtoehtoista testikokoelmaa. Lääkkeiden varsinaisten tehoaineiden ja farmaseuttisten apuaineiden silmähaitallisuutta tutkittiin viljelmissä, joilla voidaan määrittää usean kymmenen eri näytteen toksisuutta samanaikaisesti. Solut altistettiin testiaineille ja vaikutukset tutkittiin kahta eri solutoksisuustestiä käyttäen. Näistä toinen, ns. WST-1-testi, perustuu solujen mitokondrioiden toiminnan ja solujen jakaantumisen arviointiin. Laktaattidehydrogenaasitesti eli LDH-testi puolestaan osoittaa solukalvon vaurion ja kalvon läpäisevyyden muutoksen. Tutkimuksessa käytetyt, solulinjoihin perustuvat soluviljelymallit ja testimenetelmät ovat siirrettävissä laboratoriosta toiseen ja myös kaupallistettavissa helppokäyttöisiksi testiyhdistelmiksi. fi
dc.description.abstract The widely criticized Draize rabbit test is the only eye toxicity test officially accepted worldwide for regulatory purposes in the classification of slightly and moderately irritating chemicals. Today, there are no in vitro alternatives that could be used as a complete replacement for the Draize eye test. Moreover, in the current OECD guidelines, there are no tests for retinal toxicity, not even in vivo. This study was undertaken to develop corneal epithelial and retinal pigment epithelial cell culture assays as pre-screens and potential alternatives in a more comprehensive battery test for in vitro eye toxicity testing. Two basal cytotoxicity tests with effective 96-well techniques and plate readers were established to evaluate the adverse ocular effects of selected test compounds: WST-1 test as an index of mitochondrial function and cell viability/proliferation, and lactate dehydrogenase (LDH) leakage test as an index of cell membrane integrity. The confluent and post-confluent human corneal epithelial (HCE) cell line in culture medium was found to express cornea-specific cytokeratin 3, but it also expressed simple epithelium-specific cytokeratins 7, 8, 18 and 19. In most cases in the WST-1 and LDH testing, the pre-confluent HCE cell line was as sensitive to the test compounds as the pre-confluent primary cultures of rabbit corneal epithelial epithelium. The WST-1 test appeared to be an earlier indicator of toxicity than the LDH test, which also showed great variations in the cytotoxicity results. In the HCE-WST-1 interlaboratory study, the most reproducible results were obtained when the cells were exposed to the test substances for one hour in the absence of serum. The use of the human retinal pigment epithelial cell line D407 and of pig primary retinal pigment epithelial cell cultures in the WST-1 testing also yielded comparable results. In all cases, the use of serum in culture medium resulted in lower toxicity, and thus its use is not recommended. The assays studied, especially those based on cell lines, are reliable, transferable, easy-to-perform tests for ocular toxicity screening. en
dc.format.extent 102
dc.format.mimetype application/pdf
dc.language.iso en en
dc.publisher Helsinki University of Technology en
dc.publisher Teknillinen korkeakoulu fi
dc.relation.ispartofseries Technical biochemistry report / Helsinki University of Technology, Department of Chemical Technology, Laboratory of Bioprocess Engineering en
dc.relation.ispartofseries 1/2003 en
dc.relation.haspart Huhtala A., Nurmi S. K., Tähti H., Salminen L., Alajuuma P., Rantala I., Helin H. and Uusitalo H., 2003. The immunohistochemical characterisation of an SV40-immortalized human corneal epithelial cell line. Alternatives to Laboratory Animals 31, pages 409-417. [article1.pdf] © 2003 by authors and © 2003 FRAME. By permission.
dc.relation.haspart Huhtala A., Mannerström M., Alajuuma P., Nurmi S., Toimela T., Tähti H., Salminen L. and Uusitalo H., 2002. Comparison of an immortalized human corneal epithelial cell line and rabbit corneal epithelial cell culture in cytotoxicity testing. Journal of Ocular Pharmacology and Therapeutics 18, No. 2, pages 163-175. [article2.pdf] © 2002 Mary Ann Liebert, Inc. Publishers. By permission.
dc.relation.haspart Huhtala A., Alajuuma P., Burgalassi S., Chetoni P., Diehl H., Engelke M., Marselos M., Monti D., Pappas P., Saettone M. F., Salminen L., Sotiropoulou M., Tähti H., Uusitalo H. and Zorn-Kruppa M., 2003. A collaborative evaluation of the cytotoxicity of two surfactants by using the human corneal epithelial cell line and the WST-1 Test. Journal of Ocular Pharmacology and Therapeutics 19, No. 1, pages 11-21. [article3.pdf] © 2003 Mary Ann Liebert, Inc. Publishers. By permission.
dc.relation.haspart Huhtala A., Tähti H., Salminen L. and Uusitalo H., 2002. Evaluation of adverse ocular effects of 5-fluorouracil by using human corneal epithelial cell cultures. Journal of Toxicology: Cutaneous and Ocular Toxicology 21, No. 4, pages 283-292. [article4.pdf] © 2002 Marcel Dekker, Inc. By permission.
dc.relation.haspart Mannerström M., Zorn-Kruppa M., Diehl H., Engelke M., Toimela T., Mäenpää H., Huhtala A., Uusitalo H., Salminen L., Pappas P., Marselos M., Mäntylä M., Mäntylä E. and Tähti H., 2002. Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells. Toxicology In Vitro 16, pages 193-200. [article5.pdf] © 2002 Elsevier Science.
dc.subject.other Biotechnology en
dc.subject.other Medical sciences en
dc.title Corneal epithelial and retinal pigment epithelial cell culture assays as potential alternatives to animal experimentation for the evaluation of ocular toxicity en
dc.title Sarveiskalvon epiteelisoluihin ja verkkokalvon pigmenttiepiteelisoluihin perustuvat menetelmät silmätoksisuuden arvioimisessa mahdollisina eläinkokeiden vaihtoehtomenetelminä fi
dc.type G5 Artikkeliväitöskirja fi
dc.description.version reviewed en
dc.contributor.department Department of Chemical Technology en
dc.contributor.department Kemian tekniikan osasto fi
dc.subject.keyword draize eye test en
dc.subject.keyword eye irritation en
dc.subject.keyword ocular toxicity en
dc.subject.keyword alternative methods en
dc.subject.keyword in vitro test en
dc.subject.keyword cytotoxicity en
dc.subject.keyword cell culture en
dc.subject.keyword corneal epithelial cell en
dc.subject.keyword retinal pigment epithelial cell en
dc.identifier.urn urn:nbn:fi:tkk-000019
dc.type.dcmitype text en
dc.programme.major Bioprosessitekniikka fi
dc.type.ontasot Väitöskirja (artikkeli) fi
dc.type.ontasot Doctoral dissertation (article-based) en
dc.contributor.supervisor Leisola, Matti; Prof.
dc.opn Urtti, Arto; Prof.
dc.contributor.lab Laboratory of Bioprocess Engineering en
dc.contributor.lab Bioprosessitekniikan laboratorio fi
dc.rev Saari, Matti; Prof.
dc.rev Alaranta, Sakari; Dr.


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